Program Description/Abstract Adoptive cell therapy (ACT), including the use of T cells engineered to express chimeric antigen receptors (CARs), has produced unprecedented clinical outcomes and represents a new paradigm in cancer immunotherapy. However, the therapeutic efficacy is often limited by poor in vivo persistence and function of adoptively transferred T cells. How T cell fate decisions between robust effector function and long-term persistence are regulated in the tumor microenvironment (TME) remains poorly understood. Moreover, T cells undergo extensive metabolic reprograming during their activation and differentiation, but how metabolic fitness is linked to T cell function and longevity is unclear. In our preliminary studies, through an in vivo pooled CRISPR- Cas9 mutagenesis screening of metabolism-associated factors, we identified the enzyme Regnase-1 (Reg1) as a major negative regulator of antitumor CD8 T cell responses. Deletion of Reg1 resulted in drastically improved intratumoral CD8 T cell accumulation and therapeutic efficacies against mouse solid and blood cancers. Reg1- null CD8 T cells were reprogrammed in TME to acquire better persistence, survival advantage and nave/memory cell-associated gene signatures, but also retain potent effector function and enhanced mitochondrial metabolism. To explore the underlying mechanisms and additional targets, we performed a secondary in vivo genome-scale CRISPR-Cas9 screening in Reg1-null CD8 T cells and uncovered BATF as the key functional target of Reg1 in shaping mitochondrial metabolism and effector function of CD8 T cells. We also identified additional factors as putative targets for combinatorial therapy, as their co-deletion further potentiated the antitumor activity of Regnase-1-null cells. Our central hypothesis is that tumor-specific CD8 T cells can be reprogrammed to be long-lived effector T cells with adaptive metabolic and transcriptional programs, and targeting Regnase- 1 and associated pathways is an innovative strategy for cancer immunotherapy. Aim 1. Establish Reg1- BATF-dependent mitochondrial metabolism in tumor-specific CD8 T cells. Aim 2. Dissect mechanistic basis underpinning the stemness features of tumor-specific CD8 T cells. Aim 3. Explore the therapeutic potential of targeting Reg1 and combinatorial factors. We predict our studies will establish a new paradigm in understanding fundamental mechanisms of T cell fate decisions and immunometabolism and provide new avenues to improve ACT efficacy in cancer immunotherapy.